Purification and characterization of the two molecular forms of Aspergillus oryzae acid protease.

The isolation and partial characterization of the acid proteases A1 and A2 (EC3.4.23.6) from Aspergillus oryzae grown on solid bran culture are described. The purified preparations were essentially homogeneous by several criteria including sedimentation analysis and polyacrylamide gel electrophoresis. The physiochemical properties of the proteases A1 and A2 were as follows (in the order: A1, A2): molecular weight: 63 000 & 32 000; sedimentation coefficient s20, w: 3.93 and 3.16 S; diffusion constant D20, w, 5.63 - 10(-7) and 8.61 - 10(-7) CM2/S, partial specific volume, v: 0.73 ml/g for both; nitrogen content: 16.30 and 13.42%; E1% 1 cm at 280 nm: 5.9 and 11.1. The two enzymes had the same pH optima in the acid pH range, and both activated bovine pancreatic trypsinogen. The enzymes were essentially of the same amino acid composition and immunologically cross-reacted with each other. The protease A2 contained little or no carbohydrate, whereas the protease A1 was glycoprotein, containing 49% carbohydrate comprising glucose, mannose, and galactose. These results suggest that the protein portion of acid protease A1 is the same as that of acid protease A2.     

Presence and partial characterization of internal acid protease of Aspergillus oryzae.

The presence and partial characterization of the internal acid protease (EC 2.4.23.6) of Aspergillus oryzae has been investigated. Although the majority of the acid protease is external and present in the culture filtrate, a significant amount of the active enzyme is firmly bound to the cells; it is not released by repeated extraction of cells with 0.9% sodium chloride but is liberated into the soluble fraction during disruption of cells. The internal acid protease, as well as the external one, was separated into two major molecular forms (F1 and F2) with molecular weights of 60,000 and 42,000, respectively, by chromatography on Sephadex G-100 and on CM-Sephadex C-50. The partially purified internal enzymes had the same catalytic and immunological properties, as did the external enzyme.     

Purification and characterization of the two molecular forms of membrane acid protease from Aspergillus oryzae.

Two forms (M1 and M2) of the membrane-bound acid protease of Aspergillus oryzae have been purified by extraction with Triton X-100, washing with cold acetone, and repeated gel filtration on Bio-Gel A-15 m in the presence and absence of Triton X-100. The purified membrane enzymes, M1 and M2, moved as a single band in acrylamide gel electrophoresis and had apparent molecular weights of 150 000 and 60 000, respectively, as estimated by sodium dodecyl sulfate/acrylamide gel electrophoresis. These two membrane enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They immunologically cross-reacted with each other and with an extracellular acid protease from A. oryzae, and contained carbohydrate, ranging from 52.5 to 80.5% and comprising three hexoses, glucose, galactose, and mannose. While these catalytic, chemical and immunological properties are similar to those of the extracellular acid protease from A. oryzae, both membrane enzyme differed in their hydrophobic properties from external enzymes. Thus they are activated by the detergent Triton X-100 and some polar lipids.     

An acetylcholine receptor preparation lacking the 42,000 Dalton component.

Acetylcholine receptor has been purified from $\text{Electrophorus}$ in the presence of the serine protease inhibitor phenylmethyl sulfonyl flouride. The purified material has a specific toxin-binding capacity of 3.6 nmoles of toxin per mg of protein. Electrophoresis of reduced, dissociated receptor on acrylamide gels containing sodium dodecyl sulfate reveals components of 110,000, 60,000, 54,000, and 48,000 daltons. No component with an apparent molecular weight of less than 48,000 daltons is seen. Limited digestion of this preparation with trypsin results in the appearance of components of 44,000 and 42,000 daltons. Prolonged digestion with trypsin generates species with apparent molecular weights of less than 42,000 and has no effect on the specific protectable toxin-binding capacity of the preparation.     

Optimization of some additives to improve protease production under SSF

In a locally isolated Rhizopus oryzae strain highest-production of protease (388.54/g wheat bran) was observed in presence of Tween-80 and dioctyl sodium sulfosuccinate individually at 40mg/g wheat bran concentration. Under solid state fermentation biotin (0.0025mg/g wheat bran); Ca2+ (0.05mg/g wheat bran) and 1-Naphthyl acetic acid (0.01mg/g wheat bran) also showed some inducing effect on the synthesis of the enzyme protease by solid state fermentation.     

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.     

Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates.

To determine whether certain outer membrane proteins are associated with growth of Bacteroides thetaiotaomicron on polysaccharides, we developed a procedure for separating outer membranes from inner membranes by sucrose density centrifugation. Cell extracts in 10% (wt/vol) sucrose-10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4) were separated into two fractions on a two-step (37 and 70% [wt/vol]) sucrose gradient. These fractions were further resolved into outer membranes (p = 1.21 g/cm3) and inner membranes (p = 1.14 g/cm3) on sucrose gradients. About 20 to 26% of the total 3-hydroxy fatty acids from lipopolysaccharide and 2 to 3% of the total cellular succinate dehydrogenase activity were recovered in the outer membrane preparation. The inner membrane preparation contained 22 to 49% of the total succinate dehydrogenase activity and 2 to 3% of the total 3-hydroxy fatty acids from lipopolysaccharide. Outer membranes contained a lower concentration of protein (0.34 mg/mg [dry weight]) than did the inner membranes (0.68 mg/mg [dry weight]). Molecular weights of inner membrane polypeptides ranged from 11,000 to 133,000. The most prominent polypeptides had molecular weights ranging from 11,000 to 26,000. In contrast, the molecular weights of outer membrane polypeptides ranged from 17,000 to 117,000. The most prominent polypeptides had molecular weights ranging from 42,000 to 117,000. There were several polypeptides in the outer membranes of bacteria grown on polysaccharides (chondroitin sulfate, arabinogalactan, or polygalacturonic acid) which were not detected or were not as prominent in outer membranes of bacteria grown on monosaccharide components of these polysaccharides.     

Characterization of a 70S polyuridylic acid polymerase isolated from foot-and-mouth disease virus-infected cells

A polyuridylic acid polymerase complex isolated from foot-and-mouth disease virus-infected cells sedimented at 70S in a sucrose gradient and appeared in the exclusion volume of an agarose column whose molecular weight cutoff was 5 x 10(6). Phenol extraction of the complex yielded a heterogeneous band of virus-specific RNA and an apparently host cell-derived 4.5 to 5S RNA, both of which are essentially single stranded. Neither RNA served as a template in the cell-free enzyme reaction. Polyacrylamide gel analysis revealed five polypeptides with molecular weights of 50,000, 56,000, 60,000, 70,000, and 74,000 and with molar ratios of 1:2:2:1:1, respectively. Autoradiography showed P56 to be the only major virus-induced polypeptide; the other proteins are apparently of host cell origin. Electron microscopic examination suggested a cartwheel shape for the polymerase complex which was seen to dissociate as polyadenylic acid was added. Antibody previously shown to inhibit enzyme activity aggregated the 70S units.     

Mapping of three carbohydrate attachment sites in embryonic and adult forms of the neural cell adhesion molecule

The sialic-rich carbohydrate moiety of the neural cell adhesion molecule (N-CAM) undergoes major structural changes during development and plays a significant role in altering the homophilic binding of the molecule. In order to understand the mechanism of these changes, a cyanogen bromide (CNBr) fragment that contained 90% of the sialic acid of N-CAM was isolated and characterized according to the number of carbohydrate attachment sites and reactivity with specific monoclonal antibodies. The CNBr sialopeptide migrated on SDS PAGE as a broad zone of Mr 42,000-60,000. Upon treatment with neuraminidase, it was converted to a single component of Mr 42,000, and subsequent, limited treatment with endoglycosidase F gave four evenly spaced components of Mr 35,000-42,000, suggesting that it contained three attachment sites for N-linked oligosaccharides. The fragment reacted with monoclonal antibody 15G8, which detects the sialic acid in embryonic N-CAM, and with a monoclonal antibody, anti-(N-CAM) No. 2. Treatment with neuraminidase or with endoglycosidase F destroyed reactivity with 15G8 but not with anti-(N-CAM) No. 2. A similar CNBr sialopeptide was obtained from adult N-CAM; it contained sialic acid, had three N-linked oligosaccharides and reacted with anti-(N-CAM) No. 2 but not with 15G8 monoclonal antibodies. A peptide fragment, Fr2, comprising the NH2 terminal and middle regions of the molecule yielded a CNBr fragment closely similar to the fragment obtained from the whole molecule. The CNBr fragment from Fr2 reacted with monoclonal antibody anti-(N-CAM) No. 2. Fr1, comprising the NH2 terminal region alone, failed to react. These data confirm that the majority of the sialic acid is localized in the middle region of the N-CAM molecule and support the hypothesis that embryonic to adult conversion of N-CAM is the result of differences in sialidase or sialytransferase activity.     

竹叶青蛇毒丝氨酸蛋白酶的分子克隆和序列比较

利用逆转录酶与聚合酶链反应相结合的RT—PCR法,扩增出5个竹叶青(Trimeresurus stejnegeri)蛇毒丝氨酸蛋白酶的cDNAs;将扩增的cDNA片段克隆入pGEM-T载体中,筛选得到它们的基因,分别命名为TSSP-1、TSSP-2、TSSP-3、TSSP-4和TSSP-5。经末端终止法测定核苷酸序列,推导出5个丝氨酸蛋白酶的全序列;结合纯化的蛋白酶N-末端序列测定结果,推导TSSP-2、-3和-4分别编码凝血酶样酶stejnobin、纤溶酶stejnefibrase 1和2。5个丝氨酸蛋白酶分别含有1～6个N-型糖基结合位点,表明它们的计算分子量与纯化蛋白表观分子量之间的差异是由糖含量的不同造成,而其氨基酸序列相似度在60％～90％。TSSP-1和-2编码的成熟蛋白酶由236个氨基酸残基组成,TSSP-3、-4和-5的则由234个氨基酸残基组成。TSSP-1编码的蛋白酶在组成丝氨酸蛋白酶三联体催化活性中心产生了His^41-Arg^41的天然突变,这与其他自然界已发现的丝氨酸蛋白酶明显不同。     

Isolation and characterization of surface antigens from Schistosoma mansoni. I. Evaluation of techniques for radioisotope labeling of surface proteins from adult worms

Radiolabeled surface proteins of adult Schistosoma mansoni were prepared by in vitro labeling of whole worms, and by labeling freeze-thaw surface membrane extracts. Incorporation of 125I into surface proteins was attempted using the lactoperoxidase, chloramine-T, iodosulfanilic acid, and Bolton-Hunter methods. Radiolabeling of whole worms with lactoperoxidase, chloramine-T and iodosulfanilic acid yielded a single protein peak (mol wt greater than 100,000) on SDS-PAGE, and showed considerable incorporation of label in the lipid fraction. Bolton-Hunter labeling of whole worms yielded four major peaks with molecular weights of 100,000, 60,000, 30,000 and 21,000, and minor peaks with molecular weights of 26,000, 36,000, 43,000, 68,000 and 78,000; three of the four major peaks corresponded to prominent bands in Coomassie blue-stained gels. Although carbohydrate-labeling techniques were not successful, a single carbohydrate band, molecular weight greater than 100,000, was detected was PAS staining. Radiolabeling of freeze-thaw extracts yielded results similar to those obtained with whole worms. Electron microscopy revealed the tegument to be left intact and undamaged after labeling with the Bolton-Hunter reagent.     

Isolation, purification, and characterization of a new enzyme from Pseudomonas sp. M-27, carboxypeptidase G3.

A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60,000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (M(r): 15,000). The enzyme hydrolyzed predominantly acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles carboxypeptidase G1 or G2 in its glutamate-releasing activity. However, it acts not only on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G1 or G2, it was named carboxypeptidase G3.     

Evidence for and partial characterization of three major and three minor chromatographic forms of human neutrophil myeloperoxidase

Myeloperoxidase (MPO) is an enzyme found in the azurophil granules of neutrophils. Cation-exchange chromatography on carboxymethyl-cellulose previously has been used to demonstrate the heterogeneity of the peroxidase enzymes isolated from human neutrophils. In this study, fast protein liquid chromatography (FPLC) was used to separate and purify three major (I, II, and III) and three minor (IIa, IIIa, IIIb) forms of MPO from isolated neutrophil granules. Purity was confirmed by polyacrylamide gel electrophoresis in the presence of cetyltrimethylammonium bromide (CETAB-PAGE), by crossed immunoelectrophoresis, and by spectral characteristics. All three major forms were indistinguishable by immunodiffusion against rabbit antiserum, scanning spectrophotometry, and amino acid composition. They differed in their elution from a cation-exchange resin, inhibition by 3-amino-1,2,4-triazole, migration rate in CETAB-PAGE, and subunit molecular weight. Subunit molecular weight was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All three major forms appeared to consist of heavy (H), intermediate (M), and light (L) peptides. The M peptide appeared to be derived from the H subunit. All L subunits exhibited a molecular weight of 14,500. The molecular weights for the H subunits varied, and were 60,000, 59,000, and 57,000 for MPO I, II, and III, respectively. The molecular weights for the M peptides were 44,100, 43,000, and 42,000 for MPO I, II, and III, respectively. The treatment of neutrophils, granules, and extracts with protease inhibitors and sodium azide did not block the appearance of three major forms of MPO. Thus, neither protease activity nor MPO autooxidation during extraction and purification procedures is responsible for the appearance of multiple chromatographic forms of MPO derived from human neutrophils.     

Purification and characterization of glucoamylase produced by Aspergillus niger in solid state fermentation

Glucoamylases produced by Aspergillus niger grown on wheat bran in solid cultures were purified. Four different forms, GA I, GA I', GA II and GA III, were found having apparent molecular weights of 112 000, 104 000, 74 000 and 61 000 Da respectively. The enzymes are glycoproteins with a carbohydrate content of 16%, and optimal activity at 60C and pH 4.4. Activity was strongly inhibited by Hg²⁺ while Mn²⁺ and Fe²⁺ were stimulatory. The K_m values for the degradation of starch and maltose were 3.5 and 7.8 mg ml^-1, respectively.     

Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways.     

Antigenic polysaccharides of bacteria of the genus Shigella. Determination of the structure of polysaccharide chains of Shigella boydii type 9 lipopolysaccharide and detection of unusually high molecular weight glycolipid

Specific acidic polysaccharide has been isolated from the Shigella boydii type 9 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and L-rhamnose. From the results of methylation analysis, partial acid hydrolysis and 13C NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [----4)DGlcp(alpha 1----4)DGlcAp(beta 1----3)DGlcNAcp(alpha 1----3)LRhap(alpha 1----]n. The lipopolysaccharide from Sh. boydii 9 was fractionated by gel chromatography on the Sephadex G-200 column in a buffer containing sodium deoxycholate into three fractions. PAGE-SDS of the fractions obtained, 13C NMR- and chromato-mass-spectrometry data indicated that the three fractions contained the O-specific polysaccharide as the only carbohydrate component. The substance from the most high-molecular weight fraction contained unusually long O-specific chains (60,000 dalton). In the fat acid composition this fraction differed from other lipopolysaccharides by absence of beta-hydroxymyristic acid.     

Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.     

Catabolites of the third component of complement in urines of hereditary nephritis patients

Hereditary nephritis protein (HNP), an unusual urine protein from patients with hereditary nephritis (Alport Syndrome), was purified 120-fold to homogeneity. A slightly larger protein, pro-HNP, was similarly purified and was found to be a precursor of HNP. Both pro-HNP and HNP showed immunological identity to the third component of human complement, C3, and to its catabolite C3c. Pro-HNP had a molecular weight of 143,000 and, in equimolar ratio, polypeptide chains or fragments of molecular weights 75,000, 40,000, and 28,000. The largest and smallest chains contained carbohydrate. HNP had a molecular weight of 141,000 and fragments of molecular weights 60,000, 38,000, 26,000, and 17,000 in equimolar ratio; the two smallest fragments contained carbohydrate. Plasmin digestion of pro-HNP showed that the 75,000-Da chain, identical with the intact beta-chain of C3, broke down to the 60,000- and 17,000-Da fragments of HNP. In both pro-HNP and HNP, the polypeptide chains were linked by disulfide bonds, with the exception of the 17,000-Da fragment of HNP. This fragment was readily dissociated from the rest of the HNP molecule in the presence of sodium dodecyl sulfate. Amino acid analyses showed that both pro-HNP and HNP contained approximately 22 half-cystine residues per molecule. Extinction coefficients, epsilon 1% 1cm, at 280 nm were calculated to be 8.5 and 8.8 for pro-HNP and HNP, respectively.     

Production of recombinant Der fI (a major mite allergen) by Aspergillus oryzae.

Der fI is a major mite allergen. To produce Der fI by Aspergillus oryzae, we placed a DNA fragment encoding precursor-type recombinant Der fI E(-1)K (reDer fI E(-1) K), which had the C-terminal amino acid of the pro-sequence (Glu) changed to Lys, downstream of the glaA gene promoter and introduced it into Aspergillus oryzae. In liquid culture, most of the reDer fI E(-1)K produced by the transformants was degraded when culture was shaken vigorously. However, the degradation of reDer fI E(-1)K was suppressed when it was shaken gently. The processed reDer fI E(-1)K could be obtained after lysylendopeptidase and endoglycosidase Hf (Endo Hf) treatment. The yield of processed reDer fI E(-1)K was 8 mg/l. When the transformant was grown on a wheat bran culture, the yield of processed reDer fI E(-1)K reached 48 mg/kg. Because processed reDer fI E(-1)Ks obtained from both cultures had almost the same IgE-binding activity and elicited the same skin reaction as native Der fI, they could be very useful for diagnostic purposes or immunotherapy.     

Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Sequence homologies with pulmonary surfactant apoprotein and animal lectins

Phospholipase A2 inhibitor (PLI), purified from the blood plasma of the Habu snake (Trimeresurus flavoviridis), was separated into two distinct subunits, PLI-A and PLI-B. These subunits were shown to be glycoproteins with molecular weights of around 21,000-22,000. When they were deglycosylated chemically with trifluoromethanesulfonic acid, the molecular weights were found to be 17,000. Their amino acid sequences were determined by alignment of peptides obtained by lysyl endopeptidase digestion and Staphylococcus aureus V8 protease digestion. PLI-A and PLI-B were each composed of 147 amino acid residues with one residue, Asn103, being for N-linked glycosylation, and the molecular weights of their protein portions were calculated to be 16,368 and 16,408, respectively. Each subunit contained four cysteine residues, all of which exist in disulfide linkages (Cys64-Cys141 and Cys119-Cys133). The sequences of PLI-A and PLI-B showed 89.9% homology to each other. When the sequences were compared with those of lipocortins, no significant homologies were detected. But the sequences were significantly homologous to those of COOH-terminal carbohydrate recognition portions of pulmonary surfactant apoprotein and animal lectins.